Abstract 12192: MBNL1 Directly Regulates the Alternative Splicing of mRNAs That Promote Myofibroblast Differentiation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Jennifer Davis ◽  
Michelle Sargent ◽  
Jianjian Shi ◽  
Lei Wei ◽  
Maurice S Swanson ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Transforming Growth Factor ◽  
Ventricular Remodeling ◽  
Cardiac Injury ◽  
Myofibroblast Differentiation ◽  
Genome Wide ◽  
A Genome

Rationale: During the cardiac injury response fibroblasts differentiate into myofibroblasts, a cell type that enhances extracellular matrix production and facilitates ventricular remodeling. To better understand the molecular mechanisms whereby myofibroblasts are generated in the heart we performed a genome-wide screen with 18,000 cDNAs, which identified the RNA-binding protein muscleblind-like splicing regulator 1 (MBNL1), suggesting a novel association between mRNA alternative splicing and the regulation of myofibroblast differentiation. Objective: To determine the mechanism whereby MBNL1 regulates myofibroblast differentiation and the cardiac fibrotic response. Methods and Results: Confirming the results from our genome wide screen, adenoviral-mediated overexpression of MBNL1 promoted transformation of rat cardiac fibroblasts and mouse embryonic fibroblasts (MEFs) into myofibroblasts, similar to the level of conversion obtained by the profibrotic agonist transforming growth factor β (TGFβ). Antithetically, Mbnl1 -/- MEFs were refractory to TGFβ-induced myofibroblast differentiation. MBNL1 expression is induced in transforming fibroblasts in response to TGFβ and angiotensin II. These results were extended in vivo by analysis of dermal wound healing, a process dependent on myofibroblast differentiation and their proper activity. By day 6 control mice had achieved 82% skin wound closure compared with only 40% in Mbnl1 -/- mice. Moreover, Mbnl1 -/- mice had reduced survival following myocardial infarction injury due to defective fibrotic scar formation and healing. High throughput RNA sequencing (RNAseq) and RNA immunoprecipitation revealed that MBNL1 directly regulates the alternative splicing of transcripts for myofibroblast signaling factors and cytoskeletal-assembly elements. Functional analysis of these factors as mediators of MBNL1 activity is also described here. Conclusions: Collectively, our data suggest that MBNL1 coordinates myofibroblast transformation by directly mediating the alternative splicing of an array of mRNAs encoding differentiation-specific signaling transcripts, which then alter the fibroblast proteome for myofibroblast structure and function.

scholarly journals Transformer 2β Regulates the Alternative Splicing of Cell Cycle Regulator Genes to Promote Ovarian Cancer Cell Progression

2021 ◽  
Author(s):  
Peiying Fu ◽  
Ting Zhou ◽  
Dong Chen ◽  
ShiXuan Wang ◽  
Ronghua Liu
Keyword(s):  
Cell Cycle ◽  
Ovarian Cancer ◽  
Alternative Splicing ◽  
Cancer Progression ◽  
Poor Prognosis ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Level

Abstract Background: Late-stage ovarian cancer (OV) has a poor prognosis and a high metastasis rate, but the underlying molecular mechanism is ambiguous. RNA binding proteins (RBPs) play important roles in posttranscriptional regulation in the contexts of neoplasia and tumor metastasis. Results: In this study, we explored the molecular functions of a canonical RBP, TRA2B, in cancer cells. TRA2B knockdown in HeLa cells and whole-transcriptome sequencing (RNA-seq) experiments revealed that the TRA2B-regulated alternative splicing (AS) profile was tightly associated with the mitotic cell cycle, apoptosis, and several cancer pathways. Moreover, hundreds of genes were regulated by TRA2B at the expression level, and their functions were enriched in cell proliferation, cell adhesion and angiogenesis, which are related to cancer progression. We also observed that AS regulation and expression regulation occurred independently by integrating the alternatively spliced and differentially expressed genes. We then explored and validated the carcinogenic functions of TRA2B by knocking down its expression in OV cells. In vivo, a high expression level of TRA2B was associated with a poor prognosis in OV patients. Conclusions: We demonstrated the important roles of TRA2B in ovarian neoplasia and OV progression and identified the underlying molecular mechanisms, facilitating the targeted treatment of OV in the future.

scholarly journals Translational suppression via IFG-1/eIF4G confers resistance to stress-induced RNA alternative splicing in Caenorhabditis elegans

2021 ◽  
Author(s):  
Samantha C Chomyshen ◽  
Cheng-Wei Wu
Keyword(s):  
Alternative Splicing ◽  
Caenorhabditis Elegans ◽  
Rna Splicing ◽  
Molecular Mechanisms ◽  
Protein Translation ◽  
Initiation Factor ◽  
A Genome ◽  
Dividing Cells ◽  
Extended Lifespan

Splicing of pre-mRNA is an essential process for dividing cells and splicing defects have been linked to aging and various chronic diseases. Environmental stress has recently been shown to alter splicing fidelity and molecular mechanisms that protect against splicing disruption remains unclear. Using an in vivo RNA splicing reporter, we performed a genome-wide RNAi screen in Caenorhabditis elegans and found that protein translation suppression via silencing of the conserved initiation factor 4G (IFG-1/eIF4G) protects against cadmium-induced splicing disruption. Transcriptome analysis of an ifg-1 deficient mutant revealed an overall increase in splicing fidelity and resistance towards cadmium-induced alternative splicing compared to the wild-type. We found that the ifg-1 mutant up-regulates >80 RNA splicing regulatory genes that are controlled by the TGF-β transcription factor SMA-2. The extended lifespan of the ifg-1 mutant is partially reduced upon sma-2 depletion and completely nullified when core spliceosome genes including snr-1, snr-2, and uaf-2 are knocked down. Together, these data describe a molecular mechanism that provides resistance towards stress-induced alternative splicing and demonstrate an essential role for RNA homeostasis in promoting longevity in a translation-compromised mutant.

scholarly journals QKI-5 regulates the alternative splicing of cytoskeletal gene ADD3 in lung cancer

2020 ◽  
Author(s):  
Jin-Zhu Wang ◽  
Xing Fu ◽  
Zhaoyuan Fang ◽  
Hui Liu ◽  
Feng-Yang Zong ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Dependent Manner ◽  
Genome Wide ◽  
Intron Region ◽  
And Migration ◽  
Nucleotide Resolution ◽  
Anti Tumor Activity

Abstract Accumulating evidence indicates that the alternative splicing program undergoes extensive changes during cancer development and progression. The RNA-binding protein QKI-5 is frequently down-regulated and exhibits anti-tumor activity in lung cancer. However, little is known about the functional targets and regulatory mechanism of QKI-5. Here, we report that up-regulation of exon 14 inclusion of cytoskeletal gene Adducin 3 (ADD3) significantly correlates with a poor prognosis in lung cancer. QKI-5 inhibits cell proliferation and migration in part through suppressing the splicing of ADD3 exon 14. Through genome-wide mapping of QKI-5 binding sites in vivo at nucleotide resolution by iCLIP-seq analysis, we found that QKI-5 regulates alternative splicing of its target mRNAs in a binding position-dependent manner. By binding to multiple sites in an upstream intron region, QKI-5 represses the splicing of ADD3 exon 14. We also identified several QKI mutations in tumors, which cause dysregulation of the splicing of QKI targets ADD3 and NUMB. Taken together, our results reveal that QKI-mediated alternative splicing of ADD3 is a key lung cancer-associated splicing event, which underlies in part the tumor suppressor function of QKI.

scholarly journals The tumor suppressor MIR139 is silenced by POLR2M to promote AML oncogenesis

Leukemia ◽  
2021 ◽  
Author(s):  
Christiaan J. Stavast ◽  
Iris van Zuijen ◽  
Elena Karkoulia ◽  
Arman Özçelik ◽  
Antoinette van Hoven-Beijen ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Regulatory Factor ◽  
Molecular Control ◽  
Polycomb Repressive Complex 2 ◽  
Genome Wide ◽  
A Genome

AbstractMIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139 inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139 targets that mediate the tumor-suppressing activities of miR-139 in MLL-AF9 AML. We revealed that two enhancer regions control MIR139 expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139 in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139 and reveal a role for POLR2M in the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML.

scholarly journals RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽  
2021 ◽  
Author(s):  
Qiuxia Yan ◽  
Peng Zeng ◽  
Xiuqin Zhou ◽  
Xiaoying Zhao ◽  
Runqiang Chen ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Aerobic Glycolysis ◽  
Histological Grade ◽  
Migration And Invasion ◽  
Binding Motif ◽  
Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

scholarly journals Stable DNMT3L overexpression in SH-SY5Y neurons recreates a facet of the genome-wide Down syndrome DNA methylation signature

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Benjamin I. Laufer ◽  
J. Antonio Gomez ◽  
Julia M. Jianu ◽  
Janine M. LaSalle
Keyword(s):  
Dna Methylation ◽  
Down Syndrome ◽  
Neuronal Differentiation ◽  
Molecular Mechanisms ◽  
Cpg Island ◽  
Differential Methylation ◽  
Chromosome 21 ◽  
Pairwise Comparisons ◽  
Genome Wide ◽  
A Genome

Abstract Background Down syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain, and includes pan-tissue differential methylation. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. Here, we stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing (WGBS) at three different developmental phases (undifferentiated, differentiating, and differentiated). Results DNMT3L overexpression resulted in global CpG and CpG island hypermethylation as well as thousands of differentially methylated regions (DMRs). The DNMT3L DMRs were skewed towards hypermethylation and mapped to genes involved in neurodevelopment, cellular signaling, and gene regulation. Consensus DNMT3L DMRs showed that cell lines clustered by genotype and then differentiation phase, demonstrating sets of common genes affected across neuronal differentiation. The hypermethylated DNMT3L DMRs from all pairwise comparisons were enriched for regions of bivalent chromatin marked by H3K4me3 as well as differentially methylated sites from previous DS studies of diverse tissues. In contrast, the hypomethylated DNMT3L DMRs from all pairwise comparisons displayed a tissue-specific profile enriched for regions of heterochromatin marked by H3K9me3 during embryonic development. Conclusions Taken together, these results support a mechanism whereby regions of bivalent chromatin that lose H3K4me3 during neuronal differentiation are targeted by excess DNMT3L and become hypermethylated. Overall, these findings demonstrate that DNMT3L overexpression during neurodevelopment recreates a facet of the genome-wide DS DNA methylation signature by targeting known genes and gene clusters that display pan-tissue differential methylation in DS.

scholarly journals Co-Overexpression of TWIST1-CSF1 Is a Common Event in Metastatic Oral Cancer and Drives Biologically Aggressive Phenotype

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 153
Author(s):  
Sabrina Daniela da Silva ◽  
Fabio Albuquerque Marchi ◽  
Jie Su ◽  
Long Yang ◽  
Ludmila Valverde ◽  
...  
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
Inflammatory Cells ◽  
Rank Test ◽  
Mesenchymal Transition ◽  
Genome Wide ◽  
A Genome ◽  
Enhanced Expression ◽  
Advanced Stages

Invasive oral squamous cell carcinoma (OSCC) is often ulcerated and heavily infiltrated by pro-inflammatory cells. We conducted a genome-wide profiling of tissues from OSCC patients (early versus advanced stages) with 10 years follow-up. Co-amplification and co-overexpression of TWIST1, a transcriptional activator of epithelial-mesenchymal-transition (EMT), and colony-stimulating factor-1 (CSF1), a major chemotactic agent for tumor-associated macrophages (TAMs), were observed in metastatic OSCC cases. The overexpression of these markers strongly predicted poor patient survival (log-rank test, p = 0.0035 and p = 0.0219). Protein analysis confirmed the enhanced expression of TWIST1 and CSF1 in metastatic tissues. In preclinical models using OSCC cell lines, macrophages, and an in vivo matrigel plug assay, we demonstrated that TWIST1 gene overexpression induces the activation of CSF1 while TWIST1 gene silencing down-regulates CSF1 preventing OSCC invasion. Furthermore, excessive macrophage activation and polarization was observed in co-culture system involving OSCC cells overexpressing TWIST1. In summary, this study provides insight into the cooperation between TWIST1 transcription factor and CSF1 to promote OSCC invasiveness and opens up the potential therapeutic utility of currently developed antibodies and small molecules targeting cancer-associated macrophages.

Abstract 292: TRPA1 Promoting Myofibroblast Differentiation Mediate Pressure Overload-Induced Cardiac Fibrosis

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_2) ◽  
Author(s):  
Shuang Li ◽  
Dong Han ◽  
Dachun Yang
Keyword(s):  
Knockout Mice ◽  
Molecular Mechanisms ◽  
Cardiac Fibrosis ◽  
Transient Receptor Potential ◽  
Transforming Growth Factor ◽  
Ventricular Remodeling ◽  
Cardiac Fibroblasts ◽  
Gene Transfection ◽  
Pressure Overload

Background: Hypertensive ventricular remodeling is a common cause of heart failure. Activation and accumulation of cardiac fibroblasts is the key contributors to this progression. Our previous studies indicate that transient receptor potential ankyrin 1 (TRPA1), a Ca 2+ channel necessary and sufficient, play a prominent role in ventricular remodeling. However, the molecular mechanisms regulating remain poorly understood. Methods: We used TRPA1 agonists cinnamaldehyde (CA) pretreatment and TRPA1 knockout mice to understand the role of TRPA1 in ventricular remodeling of hypertensive heart. We also examine the mechanisms through gene transfection and in vitro experiments. Results: TRPA1 overexpression fully activated myofibroblast transformation, while fibroblasts lacking TRPA1 were refractory to transforming growth factor β (TGF-β) -induced transdifferentiation. TRPA1 knockout mice showed hypertensive ventricular remodeling reversal following pressure overload. We found that the TGF-β induced TRPA1 expression through calcineurin-NFAT-Dyrk1A signaling pathway via the TRPA1 promoter. Once induced, TRPA1 activates the Ca 2+ -responsive protein phosphatase calcineurin, which itself induced myofibroblast transdifferentiation. Moreover, inhibition of calcineurin prevented TRPA1-dependent transdifferentiation. Conclusion: Our study provides the first evidence that TRPA1 regulation in cardiac fibroblasts transformation in response to hypertensive stimulation. The results suggesting a comprehensive pathway for myofibroblast formation in conjunction with TGF-β, Calcineurin, NFAT and Dyrk1A. Furthermore, these data indicate that negative modulation of cardiac fibroblast TRPA1 may represent a therapeutic strategy against hypertensive cardiac remodeling.

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